The invention generally relates to novel nucleic acid molecules encoding a polypeptide or protein having the enzymatic activity of a phytase and to a method for effectively producing such a protein with phytase activity in a host cell. The invention also relates a polypeptide or protein obtained by the method according to the present invention and the use thereof as an active ingredient in the fabrication of additives for animal feed and/or for the release of phosphate of the organic material coming from the soil.
Elemental or pure phosphorus (P) is quite uncommon in nature, which is usually found as part of molecules which include the phosphate group (PO43−) joined to an organic group based on carbon of animals and plant tissues.
However, phosphorus (P) is an important nutrient for plants and animals. P is an important plant macronutrient, making up about 0.2% of a plant's dry weight. It is a component of key molecules such as nucleic acids, phospholipids, and ATP, and, consequently, plants cannot grow without a reliable supply of this nutrient. Inorganic phosphorus is the only source of phosphorous that can be absorbed and taken up by plants. Due to the only low availability of inorganic phosphorus in the soil, the farmers are forced to make available the macronutrient P.
Also animals require phosphorus as a nutrient, which can be generally provided either in the form of inorganic phosphorus, for instance, in a diet or by the degradation of the organic phosphorus various ingredients making up the diet.
The phytic acid or phytate is the major storage form of phosphorus in plants such as cereals, pulses, oilseeds, with values ranging from 1 to 5% by weight. “Phytic acid”, also referred to as inositol hexakisphosphate (IP6), or the salt thereof “phytate”, is a saturated cyclic acid. Moreover, phytic acid typically chelates and thus makes unabsorbable certain important minor minerals such as zinc and iron, and to a lesser extent, also macro minerals such as calcium and magnesium. Phytin refers specifically to the calcium or magnesium salt form of phytic acid. Catabolites of phytic acid are called lower inositol polyphosphates. Examples are inositol penta-(IP5), tetra-(IP4), and triphosphate (IP3). More than two-thirds of the phosphorus contained in the cereals and legumes are present in phytate form.
Phytate is not digestible to humans or nonruminant animals (monogastric animals), so that is is not an appropriate source of either inositol or phosphate, if eaten directly. In particular, the phosphorus and inositol present in the phytate form cannot be made bioavailable to non-ruminant animals, because they lack the digestive enzyme phytase that is required to remove phosphate from the inositol in the phytate molecule. The phosphorous present in the phytate form constitutes about between 20 and 40% of the phosphorus of vegetal origin ingested by monogastric animals.
Since phytate cannot be adsorbed by non-ruminant animals the unabsorbed phytate usually passes through the gastrointestinal tract, elevating the amount of phosphorus in the manure. Excess phosphorus excretion can lead to environmental and ecological problems, such as eutrophication. The contamination of the soil with phosphorus poses a severe problem in agriculture, especially in areas of intensive agricultural with monogastric animals.
Moreover, although some of the microorganisms in intestinal flora in the small intestine are capable of hydrolyzing phytic acid, the phosphorus is only absorbed in small amounts. On the other hand, an excess of phytate has the effect of an anti-nutritional factor since phytate is capable of chelating essential metal ions such as calcium, copper or zinc, which are necessary for animal nutrition, thereby decreasing its nutritional value.
To avoid the above mentioned drawbacks, animal food was commonly enriched with inorganic phosphorus, which in turn increased the costs of livestock production. One further approach which came up recently to alleviate these drawbacks is the direct addition of phytase, which catalyzes the hydrolysis of phytate to release and make available and accessible organic phosphorus to animal food.
The use of microbial phytase is currently approved for use in animal feed, with the purpose to increase the availability of phosphorus from phytic acid. For instance, 6-phytase produced by Aspergillus oryzae (DSM 14223) was authorized for fattening chicken, laying hens, fattening turkeys, fattening pigs and female pigs by Regulation (EC) No 255/2005 of the European Commission. These phytases are extremely weak and unstable, expressing the maximum activity of pH between 5.0 and 7.5, so that activity is substantially reduced and limited due to the low pH values in the stomach (pH 2-3). Phytase enzyme are also known to be strongly inhibited by excess of substrate (phytate) and product (inorganic phosphorus), and high temperatures (Power and Khon, 1993).
The synthetic phytases are phosphomonoesterases capable of hydrolyzing phytic acid to inorganic orthophosphate with low proportions of phosphoric esters, with pentaphosphate to monophosphate being intermediate products, and free myoinositol (Nayini and Markakis, 1986; Lasztity and Lasztity, 1988; Harland and Morris, 1995). The IUPAC-IUB (1976) has recognized the 3-phytase (EC 3.1.3.8), isolated in animals and microorganisms (Reddy and col., 1982; Lasztity and Lasztity, 1988).
Exogenous phytases have been found in microorganisms such as fungi and yeasts (e.g. Saccharomyces cerevisae and Aspergillus sp), and bacteria (Bacillus subtilis, Pseudomonas). The phytase which is obtained from Aspergillus sp follows certain order of hydrolyzing the phytate molecule, i.e. after having released the phosphate group from position 3, continue in the following order, 4, 5, 6 and 2 (Venekamp et al., 1995). These enzymes show activity at different pH 2.5 and H5.5. The activity at pH 2.5 is about 40% less effective than at pH 5.5, which is important since absorption of phosphorus occurs to a greater extent at the small intestine having a pH 5.5. (Power and Khon, 1993).
The phytase produced by Aspergillus ficuumm, is a purified glycoprotein, with an enzymatic activity that changes with temperature and pH. The optimal temperature of the enzyme is between 60 to 70° C. However, during 10 minutes at 68° C. a loss of activity to 60% was a observed (Nasi, 1990). The thermo-stable Aspergillus niger enzymes are resistant against the pelleting process and its activity is reported to be at least 5000 FTU/g. (Nasi, 1990).
WO 2011/141613 A2 describes a novel isolated gene of Serratia odorifera, which encodes a protein or polypeptide having phytase activity, a process for obtaining the same in a host yeast cell like, such as Pichia pastoris, and the use of the enzyme as an active ingredient in the fabrication of additives for animal feed. WO 2011/141613 reports on a phytase, which was not sensitive to the pH difference between the stomach (pH 3.5) and the intestinal tract (pH 7.0) and which maintained its activity over a broad pH range. The phytase described in WO 2011/1416 was reported to have improved thermo-stability.
However, the modest expression level of this Serratia phytase wild type protein still represents a bottleneck with regard to large scale protein production. There is still a strong need to identify the factors and conditions leading to an improved and effective expression of the phytase protein in a host cell. The state of the art still lacks means and methods capable of efficiently producing more protein with phytase activity per volume and over the time. Hence, there is still a strong need to improve the conditions of industrial scale production in order to provide a cost efficient and reliable method.